Compositions and methods for inducing migration by dendritic cells and an immune response

ABSTRACT

Compositions and methods of activating dendritic cells with LMP1 and LMP1-activated dendritic cell based compositions and methods are effective for dendritic cell therapy and provide an adjuvant function for vaccine administration. LMP1 or LMP1-CD40 chimeric protein may be used to activate and mature dendritic cells. LMP1 and LMP1-activated dendritic cells act as an adjuvant to enhance the cellular immune response. Also disclosed herein are kits for activating dendritic cells and for preparing a vaccine formulation. Administration of the dendritic cells transfected with LMP1 can induce an immune response against cancer or infection. The mature dendritic cells may comprise an antigen and at least one cytokine in addition to LMP1. Use of LMP1 or LMP1-CD40 provides a way to activate and mature dendritic cells that retain functional and migratory abilities without the side effects that result from maturing the dendritic cells using PGE 2 .

CROSS REFERENCE TO RELATED APPLICATIONS

This is a 371 National Stage Application of International ApplicationNo. PCT/US11/52138, filed Sep. 19, 2011, which claims priority to U.S.Provisional Application No. 61/384,779, filed Sep. 21, 2010, theentireties of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI 078834 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention relates generally to the field of immunology. Moreparticularly, the invention relates to compositions, methods, and kitsfor dendritic cell therapy.

BACKGROUND

Immune activation induces activation and maturation of dendritic cells.Dendritic cells are a part of the immune system that act asantigen-presenting cells. Dendritic cells process antigen material andpresent it on their cell surface. The antigen material may be fromviruses and bacteria.

Immature dendritic cells become activated after detecting an antigen.The antigen protein is degraded by the dendritic cell and the fragmentsare presented on the surface of the dendritic cell. After the dendriticcall is activated, it migrates to the lymph nodes and displays theantigen to other immune system cells.

Cytokine mixes currently used to activate and mature dendritic cells aresub-optimal. These mixes typically include a combination of one or moreof: interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα),interleukin-6 (IL-6), interferon-γ (IFN-γ), type I interferon,lipopolysaccharide (LPS), and prostaglandin E₂ (PGE₂). In some cases,the cytokine mix matures the dendritic cell, but does not induce themigration of the dendritic cell to the lymphoid organs where T cells areactivated. In other cases, the cytokine mix can induce migration(typically by including PGE₂), but the mix prevents important actions bythe dendritic cell, such as responsiveness to T cell help, or thecostimulatory ligand CD40L, and expression of the cytokine IL-12p70,both of which are required for optimal T cell activation and developmentof immune memory. PGE₂ is also known to induce a Th2 immune response,again limiting the effectiveness of the dendritic cells.

Traditional maturation of monocyte derived dendritic cells (MDDC)requires the addition of PGE₂. Although PGE₂ provides a mature dendriticcell that is able to home to the draining lymph node, there are manyproblems with this approach. These include making dendritic cells unableto respond to CD40 stimulation and impairing the ability of thedendritic cell to produce IL-12, a key cytokine for Th1 immuneactivation in the lymph node. (Gilboa, E., DC-based cancer vaccines. JClin Invest, 117(5): 1195-203 (2007)). Potential methods to mature MDDCwithout PGE₂ include co-transfection of molecular adjuvants such astumor necrosis family superfamily ligands (TNFSF ligands). (Kornbluth,R. S. and Stone, G. W., Immunostimulatory combinations: designing thenext generation of vaccine adjuvants. J Leukoc Biol, 80(5): 1084-102(2006); Stone, G. W., et al., Multimeric soluble CD40 ligand and GITRligand as adjuvants for human immunodeficiency virus DNA vaccines. JVirol, 80(4): 1762-72 (2006)).

There is thus a significant need for a method to activate dendriticcells while retaining the ability of the dendritic cells to migrate andthe migrated dendritic cells to induce T cell memory by secreting IL-12.

SUMMARY

Disclosed herein are compositions, kits, and methods of activatingdendritic cells with LMP1 and for preparing a vaccine formulation. Alsodisclosed herein are LMP1-activated dendritic cell based compositionsand methods that are effective for dendritic cell therapy and provide anadjuvant function for vaccine administration. Dendritic cells areactivated and matured by exposure to LMP1. Alternatively, a LMP1-CD40chimeric protein may be used to activate and mature the dendritic cells.LMP1 and LMP1-activated dendritic cells act as an adjuvant to enhancethe cellular immune response.

Genetic material such as LMP1 RNA or LMP1 DNA may be transfected intodendritic cells. Alternatively, a viral vector may be used to introduceLMP1 into the dendritic cell. A cytokine protein, e.g., IL-1β, TNFα, andIL-6, may also be incubated with the dendritic cell. Using RNA providesthe advantage that the RNA cannot integrate into the genome or bemaintained in the cell.

The mature dendritic cells may comprise an antigen and at least onecytokine in addition to LMP1. Activated, mature dendritic cells arecapable of migrating towards lymph nodes and displaying antigens toother immune system cells. Administration of the dendritic cellstransfected with LMP1 to a subject can induce an immune response againstcancer or infection. The immune response comprises secretion of IL-12 bythe dendritic cells and activation of Th1 cells.

Examples of cancers that may be treated by administration of dendriticcells transfected with LMP1 include, but are not limited to, melanoma,glioma, prostate cancer, and breast cancer. Examples of infections thatmay be treated by administration of dendritic cells transfected withLMP1 include, but are not limited to, HIV infection and hepatitis Cinfection.

Use of LMP1 or LMP1-CD40 provides a way to activate and mature dendriticcells that retain functional and migratory abilities without the sideeffects that result from maturing the dendritic cells using PGE₂.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid), and chemically-modified nucleotides. Thenucleic acid molecule may be purified. A “purified” nucleic acidmolecule is one that is substantially separated from other nucleic acidsequences in a cell or organism in which the nucleic acid naturallyoccurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% freeof contaminants). The terms include, e.g., a recombinant nucleic acidmolecule incorporated into a vector, a plasmid, a virus, or a genome ofa prokaryote or eukaryote. Examples of purified nucleic acid moleculesinclude cDNAs, fragments of genomic nucleic acid molecules, nucleic acidmolecules produced by polymerase chain reaction (PCR), nucleic acidmolecules formed by restriction enzyme treatment of genomic nucleic acidmolecules, recombinant nucleic acid molecules, and chemicallysynthesized nucleic acid molecules.

By the term “LMP1 gene,” is meant a native Epstein Barr virusLMP1-encoding nucleic acid sequence, e.g., the native Epstein Barr virusLMP1 gene; a nucleic acid having sequences from which a LMP1 cDNA can betranscribed; and/or allelic variants and homologs of the foregoing. Theterms encompass double-stranded DNA, single-stranded DNA, and RNA.

By the term “LMP1 protein,” is meant an expression product of a LMP1gene or a protein that shares at least 65% (but preferably 75, 80, 85,90, 95, 96, 97, 98, or 99%) amino acid sequence identity with theforegoing and displays a functional activity of a native LMP1 protein. A“functional activity” of a protein is any activity associated with thephysiological function of the protein.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.A “fusion protein” is a protein made by translation of an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

When referring to a peptide, oligopeptide or protein, the terms “aminoacid residue”, “amino acid” and “residue” are used interchangably and,as used herein, mean an amino acid or amino acid mimetic joinedcovalently to at least one other amino acid or amino acid mimeticthrough an amide bond or amide bond mimetic.

When referring to a nucleic acid molecule, polypeptide, or infectiouspathogen, the term “native” refers to a naturally-occurring (e.g., awild-type (WT)) nucleic acid, polypeptide, or infectious pathogen.

As used herein, the term “antigen” or “immunogen” means a molecule thatis specifically recognized and bound by an antibody.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, regions or derivativesthereof, provided by any known technique, such as, but not limited to,enzymatic cleavage, peptide synthesis or recombinant techniques.

As used herein the term “adjuvant” means any material which modulates toenhance the humoral and/or cellular immune response.

As used herein, the terms “displayed”, “presented”, or “surface exposed”are considered to be synonyms, and refer to antigens or other moleculesthat are present (e.g., accessible to immune site recognition) at theexternal surface of a structure such as a cell.

As used herein, “vaccine” includes all prophylactic and therapeuticvaccines.

As used herein, the term “biologic” refers to a wide range of medicinalproducts such as vaccines, blood and blood components, allergenics,somatic cells, genes expressing a product in gene therapy, tissues, andrecombinant therapeutic proteins created by recombinant DNA technology,antibodies, synthetic drugs, and long peptides (polypeptides), syntheticcompounds, and (glycol)proteins.

By the phrase “immune response” is meant induction of antibody and/orimmune cell-mediated responses specific against an antigen or antigensor allergen(s) or drug or biologic. The induction of an immune responsedepends on many factors, including the immunogenic constitution of thechallenged organism, the chemical composition and configuration of theantigen or allergen or drug or biologic, and the manner and period ofadministration of the antigen or allergen or drug or biologic. An immuneresponse has many facets, some of which are exhibited by the cells ofthe immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, andplasma cells). Immune system cells may participate in the immuneresponse through interaction with an antigen or allergen or other cellsof the immune system, the release of cytokines and reactivity to thosecytokines. Immune responses are generally divided into two maincategories—humoral and cell-mediated. The humoral component of theimmune response includes production of antibodies specific for anantigen or allergen or drug or biologic. The cell-mediated componentincludes the generation of delayed-type hypersensitivity and cytotoxiceffector cells against the antigen or allergen.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.

The terms “patient” “subject” and “individual” are used interchangeablyherein, and mean a mammalian subject who is to be treated, who has beentreated, or who is being considered for treatment, with human patientsbeing preferred. In some cases, the methods, kits, and compositionsdescribed herein find use in experimental animals, in veterinaryapplications, and in the development of animal models for disease,including, but not limited to, rodents including mice, rats, andhamsters, as well as non-human primates.

Although compositions, kits, and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable compositions, kits, and methods are described below.All publications, patent applications, and patents mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Theparticular embodiments discussed below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing migration of dendritic cells electroporatedwith LMP1, LMP1-CD40, or control (eGFP) RNA in the presence or absenceof cytokines or PGE₂. Migration towards a mixture of lymph nodechemokines CCL19 and CCL21 was measured.

FIG. 2 is a graph showing migration of dendritic cells incubated in thepresence of PGE₂ or alternative formulations that have been proposed toinduce migration. Migration towards a mixture of lymph node chemokinesCCL19 and CCL21 was measured.

FIG. 3 is a schematic illustration of vaccine design.

FIGS. 4A-4E show the mean fluorescence intensity (MFI) of immaturedendritic cells that were transfected with construct RNA, cultured for48 hours, stained, and analyzed by flow cytometry. Markers of maturationand activation including CCR7, CD86, CD83, CD80, and CD40 were measured.

FIGS. 5A-5F show the results of analysis of supernatant samples thatwere collected 48 hours after transfection and analyzed using the BDHuman Inflammatory Cytokine Kit. The levels of various inflammatorycytokines were measured.

FIG. 6 shows the IL-2 counts measured by an enzyme-linked immunosorbentspot (ELISpot) assay for DC cultured with peripheral blood leukocytes(PBL) and then restimulated with Gag or Ova peptide.

FIG. 7 shows the percent migration for DC added to the top well of aTranswell® support and allowed 90 minutes to migrate towards CCL19. TheTranswell® was then removed and all cells in the lower chamber werecounted.

FIG. 8 shows measurement of in vivo migration of CFSE-labeled DCtransfected with Gag mRNA or LMP1 RNA in mice injected intradermally(i.d.) with CFSE-labeled DC. LMP1 mRNA transfected DC (FIGS. 8C and 8D)were able to migrate and were detected as a distinct CFSE+ population.Control Gag treated DC did not migrate. (FIGS. 8A and 8B).

FIG. 9A shows ovary viral load 5 days after mice were vaccinated withAd5 adenoviral vector vaccines, such as Ad5-Gag+Ad5LMP1, intramuscularly(i.m.) twice at 2 week intervals and challenged two weeks later.

FIG. 9B shows lung weights after mice were vaccinated with a DNA vaccineencoding human gp100 (i.m. every 2 weeks×3) and challenged intravenously(i.v.) with B16-F10 cells and then sacrificed on day 22 post challenge.Lung weights were compared between gp100+empty vector and gp100+LMP1 orLMP1-CD40 adjuvant (4 mice in total). The dashed line is normal lungweight.

FIG. 10 shows tumor area following cell therapy with LMP1-transfecteddendritic cells. Mice were challenged with 50,000 B16-F10 cells byintradermal (i.d.) injection in the flank. Mice were either leftuntreated, injected i.d. with irradiated GVAX cells (B16 cellsexpressing GM-CSF), or injected i.d. in the footpad with bone marrowderived dendritic cells transfected with LMP1 mRNA.

FIG. 11 shows measurement of DC migration in vivo in Balb/c mice.Bone-marrow derived DC were generated by a five day culture with mGM-CSFand mIL-4. Cells were harvested and put into culture with the Mimiccytokine cocktail (FIG. 11C) or electroporated with 10 μg Gag (FIGS. 11Fand 11G) or LMP1 mRNA (FIGS. 11H and 11I). After 36 hours of maturation,cells were washed and CFSE labeled. One million CFSE labeled DC wereinjected intradermally into the flank. After 2 days, the inguinal lymphnode was dissected and processed to obtain a single cell suspension. Thesuspension was analyzed by flow cytometry and CFSE positive DC werecounted (FIGS. 11A, 11B, and 11C). Only background levels of migrationwere detected in the no treatment control (FIGS. 11D and 11E).

FIGS. 12A and 12B show that Mimic maturation induces CCR7 expression andDC migration. On day 5 non-adherent immature DC were removed fromculture and matured. CCR7 expression was determined by immuno-stainingand flow cytometric analysis 48 hours after maturation. (FIG. 12A). Themigratory ability of DCs was tested using an 8 μm pore Transwell®. (FIG.12B). 150,000 cells were allowed 90 minutes to migrate in response toCCL19 or CCL21.

FIGS. 13A and 13B show that PGE₂ causes MDDC migration and correlateswith CCR7 expression. Five days immature DC were stimulated with eitherMega-CD40L™ or LPS in the presence or absence of PGE₂. CCR7 expressionlevels were evaluated by immuno-staining and flow cytometric analysis 48hours after maturation. (FIG. 13A). The migratory ability of DCs wastested using an 8 μm pore Transwell®. (FIG. 13B). 150,000 cells wereallowed 90 minutes to migrate in response to CCL19.

FIGS. 14A and 14B show that Mimic maturation matures a migratory DC.Five day non-adherent immature DC were removed from culture and maturedusing three classic protocols found in the literature: Mimic matured,Alpha DC, and Von Gool DC. CCR7 expression was assessed by flowcytometry. (FIG. 14A). DC's migratory ability were tested using an 8 μmpore Transwell®. (FIG. 14B). 150,000 cells were allowed 90 minutes tomigrate in response to CCL19, CCL21, or CXCL12.

FIGS. 15A and 15B show that Transwell® migration of humanmonocyte-derived mRNA transfected dendritic cells were matured withvarious protocols. Day 5 non-adherent DC were electroporated with 10 ugmRNA encoding either LMP1, LMP1-CD40, or GFP control RNA. CCR7 levelswere analyzed by immuno-staining and flow cytometric analysis. RNAcontrol and LMP1 transfected cells, which received no furthermaturation, were compared to non-transfected Mimic+PGE₂ matured DC.(FIG. 15A). Migratory ability was determined using the previouslymention Transwell® migration assay in response to CCL19. (FIG. 15B).

DETAILED DESCRIPTION

Disclosed herein are methods of transfecting RNA or other geneticmaterial encoding LMP1 into ex vivo derived dendritic cells (DCs). Thesedendritic cells have been shown to migrate efficiently toward lymph nodechemokines.

The Epstein Barr Virus gene LMP1 encodes a membrane-associated proteinthat mimics the B-cell and dendritic cell activating protein CD40, orthe pattern recognition Toll Like Receptor (TLR) proteins involved ininnate immunity. Unlike CD40 or TLR, LMP1 is constitutively active,leading to the constant activation of infected B cells. Therefore, LMP1is a constitutively active analog of CD40.

Dendritic cells transfected with LMP1 can be used as an effectivereagent for dendritic cell therapy, where it will be necessary for thecells to migrate from the site of injection to the local draining lymphnode. Transfection is the process of introducing nucleic acids intocells. LMP1 can costimulate the activation of the transfected DC whileallowing the transfected DC to induce T cell memory with secretion ofIL-12 cytokines.

As disclosed herein, human ex vivo generated dendritic cellselectroporated with LMP1 and stimulated with recombinant cytokines IL-1βand TNFα efficiently migrated towards a mixture of lymph node chemokinesCCL19 and CCL21. (FIG. 1). FIG. 1 depicts migration of DC electroporatedwith LMP1, LMP1-CD40, or control (eGFP)RNA in the presence or absence ofcytokines or PGE₂. This migration can also be induced by PGE₂, but notby alternative immune stimulatory reagents. (FIG. 2). FIG. 2 depictsmigration of DC incubated in the presence of PGE₂ or alternativeformulations that have been proposed to induce migration. It is proposedthat DC transfected with LMP1 and incubated with cytokines is able toinduce IL-12p70 secretion after T cell help.

Molecular adjuvants enhance the cellular immune response. The termadjuvant includes agents that activate dendritic cells. The TNFsuperfamily of molecules such as CD40 ligand is one group of molecularadjuvant. (Kornbluth, R. S. and Stone, G. W. Immunostimulatorycombinations: designing the next generation of vaccine adjuvants. JLeukoc Biol, 80(5): 1084-102 (2006)).

Progenitor cells transform into immature dendritic cells. Immaturedendritic cells become activated after detecting an antigen that theycan present on their cell surface. Immature dendritic cells degrade theantigen protein and present the fragments on their surface.

An immune response includes activation and maturation of dendriticcells. Dendritic cells are a part of the immune system that act asantigen-presenting cells. Dendritic cells process antigen material andpresent it on their cell surface using MHC molecules. Patternrecognition receptors, such as toll-like receptors (TLR), assist thedendritic cells in detecting viruses and bacteria. After a dendriticcell is activated, it migrates to the lymph nodes. Dendritic cellsinteract with other cells within the immune system such as T cells and Bcells.

Stimulated dendritic cells produce IL-12. IL-12 helps naïve CD4+ T cellsobtain a T helper cell type 1 (Th1) phenotype. Cytokines cause thedevelopment of T helper cell type 1 (Th1) and T helper cell type 2 (Th2)cells from naïve CD4+ T cells. The Th phenotypes each produce particularcytokines and can be identified by specific cell-surface markers.

CD40 is a membrane protein found on the surface of dendritic cells,among other types of cells, and is a member of the TNF receptorsuperfamily. CD40 binds to a ligand, CD40L, which is a glycoprotein anda member of the TNF superfamily. Dendritic cells upregulate cell surfacereceptors like CD80 and CD40.

As disclosed herein, LMP1, and potentially one or more LMP1 fusionproteins, is able to induce migration of transfected DC to the localdraining lymph node at a level similar or superior to PGE₂.Prostaglandin PGE₂ induces the maturation of monocyte derived dendriticcells but may inhibit IL-12p70 by the dendritic cells. IL-12p70 is thebiologically active form of IL-12. IL-12 is produced by activateddendritic cells and promotes the development of the Th1 phenotype.Il-12p70 is a heterodimer of IL-12p40 and IL-12p35. Therefore, LMP1would be especially effective as a dendritic cell therapy reagent. Giventhat DC therapy often uses RNA encoding the antigen or immunestimulatory proteins for transfection, LMP1 can also be transfected asRNA. This is especially relevant for LMP1 because it is an oncogene. RNAis a safer method of transfection compared to DNA or viral vectors,given that RNA cannot integrate into the genome or be maintained. TheRNA is degraded over time, and therefore LMP1 encoded as RNA would notpersist in the patient after treatment.

The use of LMP1 and a LMP1-CD40 chimeric protein as vaccine adjuvants isdisclosed herein. An LMP1-CD40 chimeric protein may include thetransmembrane domain of LMP1 and the intracellular domain of CD40. LMP1and LMP1-CD40 may be used as an adjuvant for a vaccine to increase animmune response to any antigen.

An immune response may be mounted to an antigen or antigens from anypathogen as a result of vaccination against that antigen or antigens. Inone embodiment, the antigen may be derived from, but not limited to,pathogenic bacterial, fungal, or viral organisms, Streptococcus species,Candida species, Brucella species, Salmonella species, Shigella species,Pseudomonas species, Bordetella species, Clostridium species, Norwalkvirus, Bacillus anthracis, Mycobacterium tuberculosis, humanimmunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses,Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus,Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses,Plasmodium species, Trichomonas species, sexually transmitted diseaseagents, viral encephalitis agents, protozoan disease agents, fungaldisease agents, bacterial disease agents, cancer cells, or mixturesthereof.

A subject may be treated for an infectious pathogen or cancer. Examplesof infectious pathogens include viruses such as, but not limited to,influenza, HIV, dengue virus, rotavirus, HPV, HBV, HCV, CMV, HSV, HZV,and EBV, pathogenic agents including the causative agents of Malaria,Plasmodium(p) falciparum, P. malariae, P. ovale, P. vivax and P.knowlesi; the causative agent of Leishmania (L), L. major, L. tropica,L. aethiopica, L. mexicana, L. donovani, L. infantum syn. L. chagas; andpathogenic bacteria including Bacillus anthracis, Bordetella pertussis,Streptococcus pneumonia, and meningococcus.

The vaccine may be used against any cancer or with any other therapy orintervention for cancer. Examples of cancers include HPV-inducedcervical cancers (e.g., E7/E7 tumor associated antigens (TAA)), glioma,human melanoma (e.g., TRP-1, TRP-2, gp-100, MAGE-1, MAGE-3 and/or p53),breast cancer, and prostate cancer (e.g., TSA). Similarly for lungtumors, breast tumors, and leukemia, any suitable tumor associatedantigen can be used, and many have been described. Many such TAA arecommon between various cancers (e.g., CEA, MUC-1, Her2, CD20).

This invention would solve the problem of properly activating andmaturing dendritic cells for therapeutic vaccination of dendritic cellsinto patients as a treatment for cancer or chronic infections. Moreimportantly, the invention would induce the migration of these DC fromthe site of injection to the draining lymph node. This invention may beused to treat cancer and chronic infections. This invention may also beused to develop prophylactic vaccines and other immune therapiesdependent on immune activation and lymph node migration.

Disclosed herein is a different method to mature dendritic cells.Alternative technologies have been developed by individuals andcompanies that provide a mixture of cytokines and PGE₂ to maturedendritic cells and induce lymph node migration. The technologydisclosed herein provides activation of the dendritic cells and leads toincreased migration of the DC to the local draining lymph node whileretaining the ability of these migrated DC to respond to T cell help bysecreting IL-12p70.

Dendritic cell therapy or prophylactic vaccines targeting dendriticcells will induce activation and lymph node migration of the DC whilepresenting the antigen of interest to T cells in the lymph node andinducing T cell memory by secreting IL-12.

The methods disclosed herein are useful to induce the activation,migration, and IL-12 secretion by dendritic cells. Preliminary studiesshow that LMP1 is as effective as PGE₂, an inducer of dendritic cellmigration. LMP1 combined with cytokines should be an equally effectivemethod to induce migration of DC, while also being a superior method toinduce IL-12 secretion by these same DC.

LMP1 transfection appears to mature DC with greater functional abilitythan the classic Mimic matured DC. Mimic is a composition comprisingTNF-α, IL-1β, IL-6, and optionally PGE₂. After testing MDDC maturationprotocols from the literature, the only protocols that yield DC withmigratory ability are Mimic+PGE₂ and co-transfection of LMP1. Due to theincrease in cytokine expression, 2-fold higher migration, and theincreased number of Gag specific ELISpots, LMP1 appears to be a suitablereplacement for the typical PGE₂ maturation.

The below described preferred embodiments illustrate adaptations ofthese compositions, kits, and methods. Nonetheless, from the descriptionof these embodiments, other aspects of the invention can be made and/orpracticed based on the description provided below.

Biological Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (2001); and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, (1992) (with periodic updates).Immunology techniques are generally known in the art and are describedin detail in methodology treatises such as Current Protocols inImmunology, ed. Coligan et al., Greene Publishing andWiley-Interscience, New York, (1992) (with periodic updates); Advancesin Immunology, volume 93, ed. Frederick W. Alt, Academic Press,Burlington, Mass., (2007); Making and Using Antibodies: A PracticalHandbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, BocaRaton, Fla., (2006); Medical Immunology, 6th ed., edited by GabrielVirella, Informa Healthcare Press, London, England, (2007); and Harlowand Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (1988).

Nucleic acid molecules encoding an antigen as described herein may be inthe form of RNA (e.g., mRNA, microRNA, siRNA, shRNA or syntheticchemically modified RNA) or in the form of DNA (e.g., cDNA, genomic DNA,and synthetic DNA). The DNA may be double-stranded or single-stranded,and if single-stranded, may be the coding (sense) strand or non-coding(anti-sense) strand. In one embodiment, a nucleic acid can be an RNAmolecule isolated or amplified from immortalized or primary tumor celllines or dissected tumor tissue.

Typically, the subject is one who will receive a vaccine, or for whomvaccine administration is being considered.

Any suitable biological sample can be tested for immune response.Examples of biological samples include blood, serum, plasma. The samplemay be tested using any suitable protocol or assay. Examples of suitableassays include enzyme-linked immunosorbent assays (ELISAs), Westernblots, flow cytometry assays, immunofluorescence assays, qPCR,microarray analysis, etc.

In an embodiment, an antibody (e.g., monoclonal, polyclonal, Fabfragment, etc.) specific for a given protein may be used. In someembodiments, antibody binding is detected by detecting a label on theprimary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the kits, assays and methodsdescribed herein. A kit may contain antibodies and other components,packaging, instructions, or other material to aid in the use of the kit.

Described herein are kits for activating dendritic cells and forpreparing a vaccine formulation. A typical kit for activating dendriticcells as described herein may be, in one embodiment, include LMP1 andoptionally at least one cytokine A kit may include a well plate to carrythe mixture of the different reagents, as well as one or more washingbuffers. Optionally, kits may also contain one or more of the following:containers which include positive controls, containers which includenegative controls, photographs or images of representative examples ofpositive results and photographs or images of representative examples ofnegative results.

Effective Doses

The compositions described above are preferably administered to a mammal(e.g., non-human primate, bovine, canine, rodent, human) in an effectiveamount, that is, an amount capable of producing a desirable result in atreated subject (e.g., delaying or preventing onset of a disease ordisorder in the subject). Toxicity and therapeutic efficacy of thecompositions utilized in methods described herein can be determined bystandard pharmaceutical procedures. As is well known in the medical andveterinary arts, dosage for any one animal depends on many factors,including the subject's size, body surface area, age, the particularcomposition to be administered, time and route of administration,general health, and other drugs being administered concurrently.

The amount of the therapeutic agent to be administered varies dependingupon the manner of administration, the age and body weight of thepatient, and with the pathology of the disease. A composition asdescribed herein is typically administered at a dosage that activatesand matures dendritic cells, as assayed by identifying levels ofdendritic cell migration or using any that assay that measuresactivation or maturation of dendritic cells, such as IL-1α, IL-1β,IFN-α, IFN-β, IFN-γ, IL-2, IL-4, IL-6, IL-1β, IL-12, IL-15, IL-16,IL-17, IL-18, TNF-alpha. In one embodiment, the assay measuresactivation or maturation of denditric cells by measuring IFN-γ or IL-2secretion.

Therapeutic compositions described herein can be administered to asubject by any suitable delivery vehicle and route. The administrationof a composition may include a therapeutically effective amount of avaccine formulation. The composition may be provided in a dosage formthat is suitable for local or systemic administration (e.g.,parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, intracranially). In various embodiments, thecomposition may be provided in a dosage form that is suitable for oraladministration or intranasal administration. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington: The Science and Practice of Pharmacy(20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000) andEncyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C.Boylan, Marcel Dekker, New York (1988-1999)).

Compositions as described herein may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added. The composition maybe in the form of a solution, a suspension, an emulsion, an infusiondevice, or a delivery device for implantation, or it may be presented asa dry powder to be reconstituted with water or another suitable vehiclebefore use. Apart from the agent that activates and matures dendriticcells, the composition may include suitable parenterally acceptablecarriers and/or excipients. The active therapeutic agent(s) may beincorporated into microspheres, microcapsules, nanoparticles, liposomes,or the like for controlled release. Furthermore, the composition mayinclude suspending, solubilizing, stabilizing, pH-adjusting agents,tonicity adjusting agents, and/or dispersing agents.

As indicated above, the pharmaceutical compositions described herein maybe in a form suitable for sterile injection. To prepare such acomposition, the suitable active therapeutic(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution and dextrose solution.The aqueous formulation may also contain one or more preservatives(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where oneof the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

LMP1 and at least one cytokine may be mixed together in a singlecomposition, or may be administered separately. For example, the LMP1may be administered in combination with any other standard dendriticcell therapy; such methods are known to the skilled artisan anddescribed in Remington's Pharmaceutical Sciences by E. W. Martin.Formulations for oral use include a liquid containing the activeingredient(s) (e.g., LMP1 and at least one cytokine) in a mixture withnon-toxic pharmaceutically acceptable excipients.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1

Human Monocyte Isolation

PBMC were isolated from human buffy coats. Human buffy coats werepurchased from Continental Blood Bank (Miami, Fla.). Buffy coats wereficolled (GE Healthcare Ficoll) and the peripheral blood mononuclearcell (PBMC) layer was washed with warm PBS three times at decreasingspeeds: 1800 rpm, 1100 rpm, and 1000 rpm. The PBMCs were plated in aT175 flask at a concentration in 40 mL of pre-warmed human DC media(RPMI1640 (Hyclone), 5% human AB serum male only (Lonza), PenStrep (1×),and Hepes) Flasks were incubated for 2 hours at 37° C. in 5% CO₂ afterwhich all non-adherant cells were washed away with human DC media.

Monocyte Derived Dendritic Cell Preparation

Isolated monocytes were cultured for 5 days in human DC mediasupplemented with GM-CSF, 1000 U/mL (BERLEX Inc.), and IL-4, 500 U/mL(R&D systems. To harvest immature DC, flasks were placed at 4° C. for 30minutes. Non-adherent and semi-adherent dendritic cells were collectedby washing the flasks three times with cold PBS. Remaining adherentcells were discarded.

Transwell® Migration

Immature dendritic cells were resuspended in human DC media and maturedusing various protocols using Mimic cytokine mix (5 ng/mL of TNF-α[R&D], 5 ng/mL of IL-1β [R&D], 750 ng/mL of IL-6 [R&D], and 1 μg/mL ofPGE₂ [Sigma]), Mega-CD40L™ (Enzo Life Sciences), and LPS. MegaCD40L™contains two trimeric CD40 ligands linked by the collagen domain ofACRP30/adiponectin. Cells were cultured for 36 hours at 37° C. in 5%CO₂, 150,000 mature dendritic cells in 100 μL were added to 8 μmTranswell® supports (Greiner Bio-one) in triplicate. Transwell® were putinto a 24 well plate with 600 μL of human dendritic cell media and 150ng/mL of CCL19 (Peprotech). After 90 minutes incubation at 37° C.,Transwell® were removed and all dendritic cells in the lower chamber,removed with EDTA, and counted.

Flow Cytometric Analysis

Mature dendritic cells were harvested and washed once with FACS buffer(PBS+1% human AB serum+0.1% sodium azide). Cells were then blocked withblocking buffer (PBS+20% human AB serum) for 30 minutes at 4° C. Blockedcells were washed an addition time with FACS buffer and stained. FcRblocking reagent (Miltenyi Biotec) was added to all samples as anadditional blocking step. Surface CCR7 was stained using Anti-CCR7PE-Cy7(BD Bioscience). Stained samples were run on a LSR-Fortessa (BDBioscience).

mRNA Preparation and Dendritic Cell Transfection

Sequences encoding pLyso-Gag, LMP1, and LMP1-CD40 were cloned into thepGEM4z plasmid containing a T7 promoter and a 64 base pair polyA tail(pGEM4Z/A64). pGEM4Z containing GFP (provided by Eli Gilboa) was used asa control in all human transfection experiments. Ambion mMESSAGEmMACHINE RNA in vitro transcription kit was used for in vitro mRNAsynthesis.

Five day immature dendritic cells were harvested and washed two timeswith Opti-MEM medium (GIBCO). Cells were resuspended in Opti-MEM at aconcentration of 1×10⁷ mL. 200 μL of cells was mixed with 10 μg of RNAand transfected in a pre-chilled 0.4 cm electroporation cuvette(Bio-Rad). A Bio-Rad Gene Pulser X cell Electroporator was used toelectroporate the dendritic cells at 350 V and 150 μF. Postelectroporation, DC were cultured in 6-well plates in the presence ofGM-CSF and IL-4 and maturation cocktails where indicated. After 48hours, migration was tested by Transwell® migration assay as describedherein.

Preparation of Mouse Bone Marrow-Derived DC

Bone marrow was harvested from the femurs of 6-7 week old Balb/c mice byflushing each bone shaft with cold PBS. The PBS marrow solution was thenstrained using a 70 μm cell strainer. Any large marrow pieces werehomogenized using the plunger of a 3 mL syringe. Cells were washed twicewith PBS and resuspended at a concentration of 1×10⁶ cell/mL in R10media (RPMI1640+10% FBS) containing 20 ng/mL murine GM-CSF (Peprotech)and 10 ng/mL of murine IL-4 (Peprotech). Cells were plated in 6 wellplates and incubated from 5 days at 37° C. in 5% CO₂ (day 0). On day 3,media was changed and 3 mL of fresh complete R10 medium containingGM-CSF and IL-4 were added. On day 5, the non-adherent cells werecollected by washing three times with cold PBS.

Bone Marrow Derived Dendritic Cell (BMDDC) Transfection

The immature dendritic cells removed from the 6 well plate and washedtwice with Opti-MEM medium and resuspended in Opti-MEM at aconcentration of 1×10⁷ cells/mL. Dendritic cells were mixed with 10 μgof RNA and were electroporated in a chilled 0.4 cm electroporationcuvette at 350 V and 150 μF. After transfection, cells were replated inR10 media containing GM-CSF and IL-4 and maturation cocktails whereindicated.

CFSE Labeling

On day 7 (untransfected) or 48 hours after electroporation, non adherentand loosely adherent cells were harvested. The cells were resuspended ina 15 mL tube in a 1 mL volume of PBS containing 5% FBS. 110 μL of PBSwas added horizontally as a droplet to the non-wetted portion of thetube. 1.1 μL of 5 mM CFSE was added to this droplet, and the tube wasimmediately capped, inverted, and vortexed. After thorough mixing, tubeswere incubated in the dark for 5 minutes at room temperature. The cellswere then quenched with ten volumes of cold R10 media and kept in icefor 5 minutes. Labeled cells were washed twice with cold R10 media andfollowed by two additional washes with PBS.

In Vivo Migration Assay

The CFSE-labeled cells were injected intradermally into the thighs ofBalb/c mice (1-4 million cells injected per mouse). The no treatmentmouse was injected with identical volumes of PBS. After 48 hours,inguinal lymph nodes was harvested. Lymph node were cut into pieces andincubated for 25 minutes with Collagenase Type II (200 U/mL) and 200U/mL DNase. EDTA was then added to 0.01 M and incubated for anadditional 5 minutes. The lymph node was then ground through a 70 μmcell strainer and washed thoroughly. The cells were spun down,transferred to FACS tubes, fixed using 2% formalin, and run on anLSR-Fortessa (BD Bioscience) to obtain an accurate number ofsuccessfully migrated CFSE labeled dendritic cells.

DC Maturation Protocols

Immature dendritic cells were resuspended in human DC media and maturedusing various protocols including Mimic, Alpha, and Von Gool. Theprotocols are as follows: (1) Mimic matured cytokine mix (5 ng/mL ofTNF-α [R&D)], 5 ng/mL of IL-1β [R&D], 750 ng/mL of IL-6 [R&D], and 1μg/mL of PGE₂ [Sigma]), (2) Alpha DC cytokine mix (3000 U/ml TNF-α, 1000U/ml IFN-γ, and 20 μg/ml polyinosinic:polycytidylic acid (Poly I:C)), or(3) Von Gool DC cytokine mix (2000 U/ml IL-1β and 1000 U/ml TNF-α). Thecells were cultured for 36 hours at 37° C. in 5% CO₂ and then used formigration assays.

Example 2

Disclosed herein is a method to mature dendritic cells that retainfunctional and migratory abilities without the side effects that resultfrom maturing the dendritic cells using PGE₂. FIG. 3 is a schematicillustration of vaccine design. The DC may be matured using LMP1 orLMP1-CD40 and optionally at least one cytokine instead of Mimic.

Vaccines were prepared by the following method. Monocytes are removedfrom the patients and matured into immature dendritic cells using GM-CSFand IL-4. The immature DC are electroporated with antigen mRNA andmatured with Mimic. Mimic is a composition comprising TNF-α, IL-1β,IL-6, and PGE₂. Specifically, the Mimic cytokine mix may be comprised of5 ng/ml TNFα, 5 ng/ml IL-1β, 750 ng/ml IL-6 and 1 μg/ml PGE₂. The DCwere cultured overnight at 37° C. in 5% CO₂. Matured DC that areexpressing antigen are cryopreserved and readministered to the patientover the course of their treatment. (FIG. 3).

Molecular adjuvants were transfected as mRNA along with HIV Gag mRNA byelectroporation. After 48 hours of maturation, supernatants werecollected and measured for cytokine levels by cytometric bead array.Maturation and activation markers were analyzed by cell surface stainingand flow cytometric analysis. Mature antigen loaded DC were culturedwith PBL for 12 days followed by re-stimulation with 10 μg/ml Gagpeptide and ELISpot for IFN-γ and IL-2. DC migratory ability was testedby Transwell® migration assay in response to CCL19.

Example 3

Referring to FIGS. 4A-4E, immature dendritic cells were transfected with10 μg construct RNA and cultured for 48 hours. DC were then stained andanalyzed by flow cytometry.

LMP1 and LMP1-CD40 significantly increase markers of maturation andactivation including CCR7, CD86, CD83, CD80, and CD40. (FIGS. 4A-4E).LMP1-CD40 consists of the LMP1 transmembrane domain and the CD40cytoplasmic tail domain. The N-terminal residues of LMP1 form sixtransmembrane regions. The cytoplasmic tail contains signaling domains.

CCR7 is upregulated to levels comparable to those of Mimic matured DC,which suggests that the DC may be able to migrate to the draining lymphnode. (FIG. 4A).

Example 4

Referring to FIGS. 5A-5F, supernatant samples were collected 48 hoursafter transfection and analyzed using the BD Human Inflammatory CytokineKit. The BD Human Inflammatory Cytokine Kit can be used toquantitatively measure interleukin-8 (IL-8), interleukin-β (IL-β),interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor(TNF), and interleukin-12p70 (IL-12p70) protein levels. (BD CytometricBead Array (CBA) Human Inflammatory Cytokines Kit Instruction Manual,Becton, Dickinson, and Company, (2010). The kit includes beads of knownsize and fluorescence that capture a particular analyte so that thepresence of the analyte can be detected using flow cytometry. Thephycoerythrin (PE)-conjugated antibodies present in the kit act as adetection reagent with a fluorescent signal proportional to the amountof bound analyte.

LMP1 and LMP1-CD40 significantly up-regulate the expression ofinflammatory cytokines (FIGS. 5A-5F). The cytokine profile appears tomirror what is seen for Mimic DC. Expression of IL-12p70 in the culturesupernatant was not detected. (FIG. 5D).

Example 5

Referring to FIG. 6, DC were cultured with PBL for 12 days. On day 12,the DC were restimulated with Gag or Ovalbumin (Ova) peptide andmeasured for IFN-γ and IL-2 secretion by an enzyme-linked immunosorbentspot (ELISpot) assay. The Gag and Ova peptides act as antigens. Gagpeptides may be used to determine if matured DC loaded with Gag peptidesare able to induce Gag specific IFN-γ and IL-2 responses. Ova peptidesare used for the study of CD4 T cell response. Class II MHC binds theOva peptides and presents them to T cells.

The results of the IFN-γ and IL-2 ELISpot assays show a significantincrease in the ability of DCs to present antigen and activate T cellswhen co-transfected with LMP1. (FIG. 6). Levels were similar to thosefor DCs matured by Mimic.

Example 6

Referring to FIG. 7, 150,000 DC were added to the top well of apermeable support (in this example, a Transwell® permeable support). TheDC were allowed 90 minutes to migrate towards CCL19 after which theTranswell® was removed and all cells in the lower chamber were counted.CCL19 is a chemokine that attracts dendritic cells and binds to the CCR7receptor.

LMP1 and LMP1-CD40 alone failed to mature DC capable of migration. (FIG.7). LMP1 transfected DC cultured in the presence of Mimic without PGE₂showed 2-fold higher migratory abilities than Mimic matured DC+PGE₂.LMP1-CD40 transfected DC in the presence of Mimic migrate as well asPGE₂ matured DC.

Example 7

Referring to FIG. 8A-8D, in vivo migration of DC can be measured in miceinjected intradermally (i.d.) with carboxyfluorescein succinimidyl ester(CFSE)-labeled DC. CFSE is a fluorescent dye that can be used to labellymphocytes and track their migration in vivo. A million bone marrowderived BALB/c DC were labeled with CFSE and injected i.d. into theflank of mice. After 24 hours, the draining inguinal lymph node wasdissected and measured for CFSE.

LMP1 mRNA transfected DC (FIGS. 8C and 8D) were able to migrate and weredetected as a distinct CFSE+ population. Control Gag treated DC did notmigrate. (FIGS. 8A and 8B).

Example 8

Referring to FIG. 9, pilot studies were performed evaluating LMP1 andLMP1-CD40 as vaccine adjuvants. In FIG. 9A, Ad5 adenoviral vectorvaccines combining Ad5-Gag+Ad5-LMP1 induced a 1-2 log decrease invaccinia-Gag viral load after murine challenge when compared toAd5-Gag+Ad5-GFP control virus. Mice were vaccinated intramuscularly(i.m.) twice at 2 week intervals, then challenged two weeks later. Ovaryviral load was measured 5 days later.

In FIG. 9B, mice vaccinated with a DNA vaccine encoding human gp100(i.m. every 2 weeks×3) were challenged intravenously (i.v.) with B16-F10cells and then sacrificed on day 22 post challenge. Lung weights werecompared between gp100+empty vector and gp100+LMP1 or LMP1-CD40 adjuvant(4 mice in total). All lungs with increased weight had visible tumors.The dashed line is normal lung weight.

Example 9

Referring to FIG. 10, cell therapy with LMP1-transfected dendritic cellsreduce tumor growth in C57BL/6 tumor bearing mice at a similar level tothe cancer therapy GVAX.

Mice were challenged with 50,000 B16-F10 cells by intradermal (i.d.)injection in the flank. Mice were either left untreated, injected i.d.with irradiated GVAX cells (B16 cells expressing GM-CSF), or injectedi.d. in the footpad with bone marrow derived dendritic cells transfectedwith LMP1 mRNA.

Example 10

Referring to FIGS. 11A-11I, LMP1 induces DC migration in vivo in Balb/cmice. Bone-marrow derived DC were generated by a five day culture withmGM-CSF and mIL-4. Cells were harvested and either put into culture withthe Mimic cytokine cocktail (FIG. 11C) or electroporated with 10 μg GagmRNA (FIGS. 11F and 11G) or 10 μg LMP1 mRNA (FIGS. 11H and 11I). After36 hours of maturation, cells were washed and CFSE labeled. One millionCFSE labeled DC were injected intradermally into the flank. After 2days, the inguinal lymph node was dissected and processed to obtain asingle cell suspension. The suspension was analyzed by flow cytometryand the CFSE positive DC were counted. (FIGS. 11A, 11B, and 11C). Thenegative PBS control reported minimal background levels. (FIG. 11A).Unlike in human DC, immature BMDC do possess migratory abilities. WhenDC were matured with Mimic+PGE₂, these levels were 2.5 times greater,suggesting that maturation still increases migration rates. (FIG. 11C).This increase in migration is consistent with observations in human DC.

LMP1 increases migration rates in mouse bone marrow-derived dendriticcells in the absence of PGE₂. In order to determine if LMP1 increasesmouse BMDC migration rates as it does in humans, immature BMDC weretransfected with mRNA encoding LMP1 or Gag as a RNA control treatment.These DC were then matured in Mimic cocktail without PGE₂ and injectedinto the mice. After 48 hours, migrated DC were counted in the inguinallymph nodes.

Only background levels of migration were detected in the no treatmentcontrol. (FIGS. 11D and 11E). These levels were identical to the Gagtransfected DC control suggesting that RNA transfection alone does notinduce DC migration. (FIGS. 11F and 11G). LMP1 transfection did increasemigration rates. (FIGS. 11H and 11I). A distinct population of cells isvisible when run on a flow cytometer. Although these migration rates aresignificantly less than what is seen in vitro, these results stillsuggest that LMP1 does indeed increase the functional ability of DC tomigrate to a draining lymph node.

Example 11

Referring to FIGS. 12A and 12B, Mimic maturation induces CCR7 expressionand DC migration. Monocyte derived dendritic cells were generated asfollows: Isolated monocytes were cultured for 5 days in human DC mediasupplemented with GM-CSF, 1000 U/mL (BERLEX Inc.), and IL-4, 500 U/mL(R&D Systems). To harvest immature DC, the flasks were placed at 4° C.for 30 minutes. Non-adherent and semi-adherent dendritic cells werecollected by washing the flasks three times with cold PBS. The remainingadherent cells were discarded.

On day 5 non-adherent immature DC were removed from culture and matured.CCR7 expression was determined by immuno-staining and flow cytometricanalysis 48 hours after maturation. (FIG. 12A). DC's migratory abilitywere tested using an 8 μm pore Transwell®. (FIG. 12B). 150,000 cellswere allowed 90 minutes to migrate in response to CCL19 or CCL21.

Dendritic cell maturation with PGE₂ induced CCR7 expression andmigration in human MDDC. PGE₂ has been known to cause various sideeffects in MDDC. Therefore, it was studied whether immature DC possessthe same ability to migrate as Mimic matured DC. Surface expression ofCCR7 was studied to see if maturation upregulated a key chemokinereceptor necessary for migration to the draining lymph node. (FIG. 12A).Immature DC were matured for 36 hours in the presence of Mimic and PGE₂,then stained from CCR7 expression and analyzed by flow cytometry. Mimicmaturation with PGE₂ significantly increases surface expression of CCR7,suggesting better rates of migration.

To test migration, the same cells were give 90 minutes to migrate toeither CCL19 or CCL21 (chemokines for CCR7) in the Transwell® migrationassay. Immature DC showed low levels of migration, around 5%, to bothCCL19 and CCL21. (FIG. 12B). As expected, Mimic matured DC migrated toCCL19 and CCL21 at a 4 fold higher rate. This suggested that maturationis key for DC migration.

Example 12

Referring to FIGS. 13A and 13B, PGE₂ is necessary for MDDC migration andcorrelates with CCR7 expression. Five days immature DC were stimulatedwith either Mega-CD40L™ or LPS in the presence or absence of PGE₂. CCR7expression levels were evaluated by immuno-staining and flow cytometricanalysis 48 hours after maturation. (FIG. 13A). The migratory ability ofDCs was tested using an 8 μm pore Transwell®. (FIG. 13B). 150,000 cellswere allowed 90 minutes to migrate in response to CCL19.

PGE₂ induces in vitro DC migration. Since maturation appeared to be animportant part of DC migration, it was studied whether there was asuitable replacement for PGE₂ that would allow the same functionalbenefits, while eliminating some of the negative side effects. Five dayimmature monocyte-derived dendritic cells were matured with acombination of immunostimulants including Mimic, PGE₂, Mega-CD40L™, andLPS. After 48 hours, DC were harvested and stained for CCR7 expressionand analyzed by flow cytometry.

Forms of maturation that did not include PGE₂ failed to increase levelsof surface CCR7 expression (FIG. 13A). These protocols expression levelswere similar to the basal level of expression seen on immature DC.However, the addition of PGE₂ to the maturation cocktail significantlyincreased CCR7 levels. This expression level seemed to be additive, inthat the more immunostimulants added, the higher the level of CCR7expression. (FIG. 13A).

PGE₂ induces dendritic cell migration in vitro. In vitro migration wastested to see if levels of CCR7 expression correlate to the functionalability of DCs to migrate to chemokines Migration was tested using aTranswell® migration assay to CCL19.

In the absence of any maturation cocktail, DC migration is negligible(below 5%). Similar to observations in CCR7 expression, any maturationprotocol that did not contain PGE₂ yielded a DC that was incapable ofmigrating to CCL19. (FIG. 13B). However, after the addition of PGE₂ tothe cocktail, DC maturation was significantly increased (20-35%).Interestingly, the addition of Mega-CD40L™ to Mimic+PGE₂ actuallydecreases migration when compared to Mimic+PGE₂ alone, similar to thedecrease that was seen in CCR7 expression. The addition of LPS alongwith Mimic and PGE₂ yielded the best migration (31%). The addition ofMega-CD40L to this mixture had very minimal benefits only increasing themigration by 1%.

Example 13

Referring to FIGS. 14A and 14B, the Mimic maturation protocol matured amigratory DC. Five day non-adherent immature DC were removed fromculture and matured using three classic protocols found in theliterature: Mimic matured, Alpha DC, and Von Gool DC. The Mimic matured,Alpha DC, and Von Gool DC maturation protocols are as follows: (1) Mimicmatured cytokine mix (5 ng/mL of TNF-α [R&D)], 5 ng/mL of IL-1β [R&D],750 ng/mL of IL-6 [R&D], and 1 μg/mL of PGE₂ [Sigma]), (2) Alpha DCcytokine mix (3000 U/ml TNF-α, 1000 U/ml IFN-γ, and 20 μg/mlpolyinosinic:polycytidylic acid (Poly I:C)), or (3) Von Gool DC cytokinemix (2000 U/ml IL-1β and 1000 U/ml TNF-α). The cells were cultured for36 hours at 37° C. in 5% CO₂ and then used for migration assays.

CCR7 expression was assessed by flow cytometry. (FIG. 14A). Themigratory ability of the DCs was tested using an 8 μm pore Transwell®.(FIG. 14B). 150,000 cells were allowed 90 minutes to migrate in responseto CCL19, CCL21, or CXCL12.

Mimic-matured dendritic cells express the highest amounts of CCR7compared to common maturation protocols. It appears that PGE₂ isnecessary to induce CCR7 expression and DC migration. Common maturationprotocols that do not include PGE₂ were tested to see if migration couldbe induced without the use of PGE₂. Three common maturation protocolswere used to mature DC: Mimic matured, Alpha DC, and Von Gool DC. Aftermaturation, cells were stained with CCR7 and analyzed by flow cytometry(FIG. 14A). The Von Gool protocol failed to induce any CCR7 expressionover normal levels on immature DC. The Alpha DC protocol induced modestCCR7 expression but levels were much lower than the classic Mimicmatured DC. (FIG. 14A).

Mimic maturation induces the best migration rates in MDDC. All threecommon protocols were tested against CCL19, CCL21, and CXCL12 todetermine if certain protocols gave preferential migration to certainchemokines (FIG. 14B). Mimic maturation yielded the highest migrationrates to CCL19 (56%). Mimic DC also migrated to both CCL21 and CXCL12but at a much lower levels than CCL19 (−22%). Although Alpha DC showedmodest CCR7 expression, migration rates did not correlate. Levels werecomparable to that of immature DC. DC matured using the Von Goolprotocol did not migrate to any chemokine (FIG. 14B).

Example 14

Referring to FIGS. 15A and 15B, Transwell® migration of humanmonocyte-derived mRNA transfected dendritic cells were matured withvarious protocols. Day 5 non-adherent DC were electroporated with 10 μgmRNA encoding either LMP1, LMP1-CD40, or GFP control RNA. CCR7 levelswere analyzed by immuno-staining and flow cytometric analysis. RNAcontrol and LMP1 transfected cells, which received no furthermaturation, were compared to non-transfected Mimic+PGE₂ matured DC.(FIG. 15A). Migratory ability was determined using the Transwell®migration assay in response to CCL19. (FIG. 15B).

LMP1 induced CCR7 expression. LMP1 also induced migration levels greaterthan that of Mimic matured DC. Immature human dendritic cells obtainedfrom buffy coats were transfected with mRNA encoding molecular adjuvantsLMP1 or LMP1-CD40. After 48 hours, CCR7 levels were measured by flowcytometry. (FIG. 15A). LMP1-transfected DC significantly upregulatedCCR7 to levels similar to those of DC matured with Mimic+PGE₂. Thisfinding suggested that LMP1 may give DC the functional ability tomigrate without the use of PGE₂. To test this, DC were transfected andmatured with or without Mimic. (FIG. 15B). Interestingly, LMP1 andLMP1-CD40 transfected DC could not migrate to CCL19 even though theyexpress high levels of CCR7. When transfected DC were matured with Mimicwithout PGE₂, migration was achieved. LMP1-CD40 transfected DC+Mimicmigrated as well as Mimic+PGE₂ DC. When LMP1 transfected DC were maturedwith Mimic without PGE2, they achieved migration rate almost twice thatof Mimic+PGE₂. These data suggest that LMP1 may be a superiorreplacement for PGE₂ when maturing monocyte-derived dendritic cells.

Other Embodiments

Any improvement may be made in part or all of the compositions, kits,and method steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. This invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contraindicated by context.

REFERENCES

-   Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic    Press, Burlington, Mass., (2007)-   BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit    Instruction Manual, Becton, Dickinson, and Company, (2010).-   Current Protocols in Immunology, ed. Coligan et al., Greene    Publishing and Wiley-Interscience, New York, (1992) (with periodic    updates).-   Current Protocols in Molecular Biology, ed. Ausubel et al., Greene    Publishing and Wiley-Interscience, New York, (1992) (with periodic    updates).-   Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick    and J. C. Boylan, Marcel Dekker, New York (1988-1999)).-   Gilboa, E., DC-based cancer vaccines. J Clin Invest, 117(5):    1195-203 (2007)-   Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., (1988).-   Kornbluth, R. S. and Stone, G. W., Immunostimulatory combinations:    designing the next generation of vaccine adjuvants. J Leukoc Biol,    80(5): 1084-102 (2006).-   Lu, W., et al., Therapeutic dendritic-cell vaccine for chronic HIV-1    infection. Nat Med,. 10(12): 1359-65 (2004).-   Making and Using Antibodies: A Practical Handbook, eds. Gary C.    Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fla., (2006)-   Medical Immunology, 6th ed., edited by Gabriel Virella, Informa    Healthcare Press, London, England, (2007)-   Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed.    Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y., (2001).-   Remington: The Science and Practice of Pharmacy (20th ed.),    ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2000).-   Stone, G. W. et al., Multimeric soluble CD40 ligand and GITR ligand    as adjuvants for human immunodeficiency virus DNA vaccines. J Virol,    80(4): 1762-72 (2006).

What is claimed is:
 1. A composition comprising dendritic cellstransfected with a nucleic acid sequence encoding a full-length latentmembrane protein (LMP1) protein, wherein the dendritic cells are capableof migrating towards lymph node chemokines, secreting IL-12 andactivating T cells.
 2. The composition of claim 1, wherein the nucleicacid is RNA.
 3. The composition of claim 1, further comprising a mediaformulation comprising at least one cytokine selected from the groupconsisting of: IL-1β, TNFα, and IL-6.
 4. The composition of claim 3,wherein the at least one cytokine consists of: IL-1β, TNFα, and IL-6. 5.The composition of claim 1, wherein the dendritic cells are humandendritic cells.
 6. A composition for inducing dendritic cell migrationcomprising: (i) a full-length LMP1-CD40 chimeric protein or a nucleicacid sequence encoding a full-length LMP1-CD40 chimeric protein, (ii)IL-1β, TNFα and IL-6, wherein dendritic cells that are contacted withsaid composition are capable of migrating towards lymph node chemokines,secreting IL-12 and activating T cells.
 7. The composition of claim 6,wherein the composition is a vaccine adjuvant for treating a disease orcondition in a subject, and further comprises a pharmaceuticallyacceptable excipient.
 8. A composition comprising dendritic cellstransfected with a nucleic acid sequence encoding a full-lengthLMP1-CD40 chimeric protein, wherein the dendritic cells are capable ofmigrating towards lymph node chemokines, secreting IL-12 and activatingT cells.